Analysis of estrogenic effects by quantification of green fluorescent protein in juvenile fish of a transgenic medaka

The ChgH-GFP strain of the teleost medaka contains a regulatory region of the estrogen-responsive choriogenin H (chgH) gene fused to the green fluorescent protein (GFP) gene. The strain was developed for the identification of environmental estrogens by noninvasive analysis of GFP fluorescence. In the present study, a quantification method for GFP by image analysis was established and applied to the analysis of time- and concentration-dependent GFP fluorescence in juvenile fish. Concentration-response analyses were performed with fish exposed for 14 d to 17β-estradiol (0.37-367 pM), genistein (0.37-367 nM), or p-nonylphenol (0.367-1,835 nM). By means of image analysis, it was shown that ChgH-GFP was induced at 183.5 pM or greater 17β-estradiol. Time-course and recovery experiments indicated a strong accumulation of GFP in the liver. Results of reverse transcriptase-polymerase chain reaction analysis of choriogenin H and vitellogenin demonstrated induction of gene expression for the same range of concentrations as that for GFP analysis. Neither expression of these genes nor GFP fluorescence was induced by genistein and p-nonylphenol. Although the ChgH-GFP strain failed to detect these weakly estrogenic compounds, the simplicity of the GFP quantification during early life stages of fish offers promising possibilities for further developments of transgenic strains using different target regulatory sequences.